Process for making iron oxide nanoparticle preparations for cancer hyperthermia

ABSTRACT

Iron oxide nanoparticle compositions, methods of preparing the nanoparticles using high gravity controlled precipitation (HGCP), and methods of using the nanoparticles are disclosed.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/648,080, filed May 28, 2015, now U.S. Pat. No. 10,406,228, which is a§ 371 U.S. National Entry Application of International PatentApplication PCT/US2013/072328, filed Nov. 27, 2013, which claims thebenefit of U.S. Provisional Application No. 61/793,871, filed Mar. 15,2013, and U.S. Provisional Application No. 61/731,192, filed Nov. 29,2012, the content of each of the aforementioned applications is hereinincorporated by reference in its entirety.

BACKGROUND

Despite the great promise, magnetic nanoparticle hyperthermia (mNHP) hashad limited success in clinical applications. This limited success isdue, in part, to technical difficulties of selective heat delivery tothe target tissue without overheating adjacent normal tissue. Magneticnanoparticle hyperthermia for cancer therapy is an application ofalternating magnetic fields (AMFs) in which magnetic nanoparticleheating depends upon both AMF frequency and amplitude (Jordan et al.,1997; Rosensweig, 2002; Bordelon et al., 2011). Generally, the objectiveis to develop nanoparticle and AMF device combinations that produce amaximum particle-associated heating rate, or loss power for a given flux(peak-to-peak) magnetic field. For many magnetic materials, the losspower increases both with increasing AMF frequency and amplitude, thusmotivating development of particles that generate therapeutic heatingwith safe AMF exposure. For a given magnetic ion oxide nanoparticle(MION) formulation localized in tissue, the amount of heat depositedduring mNHP depends on both the intratumoral iron-oxide nanoparticle(IONP) concentration and AMF parameters.

When a region of tissue in an animal or a patient is subjected toalternating magnetic field (AMF), non-specific Joule heat is depositedinto the tissue due to eddy currents. The total non-specific powerdeposited is proportional to H²f²r²; where H and f are AMF amplitude andfrequency, and r is the radius of the eddy current path, which isrelated to the radius of tissue exposed to AMF. For most iron-oxidenanoparticles (IONPs) the heat generating ability is proportional to HY.Hence, lower AMF frequencies in the range of 100 kHz to 400 kHz aretypically used in mNPH applications (Atkinson et al., 1984). For mNPH tobe effective, the IONPs should generate higher heating at low fieldamplitude, or H-values.

SUMMARY

In some aspects, the presently disclosed subject matter provides aprocess for preparing one or more surfactant-coated magnetic metal oxideparticles, the process comprising: (a) providing a salt solution of ametal; (b) contacting the salt solution of the metal with a precipitantsolution to form a reactant solution; (c) rapidly micro-mixing thereactant solution to initiate formation of metal oxide crystals undercontrolled nucleation conditions; (d) continuing to rapidly micro-mixthe reactant solution under high gravity conditions to control crystalgrowth of one or more metal oxide particles formed therein; (e) coatingthe one or more metal oxide particles with a surfactant; (0 separatingthe one or more coated metal oxide particles from the reactant solutionand one or more by-products, if present, formed therein; and (g)exposing the one or more coated metal oxide particles to hightemperature and high pressure in an inert gas environment for a periodof time to form one or more surfactant-coated magnetic metal oxideparticles.

In other aspects, the presently disclosed subject matter provides one ormore surfactant-coated magnetic metal oxide particles prepared by thepresently disclosed methods.

In more particular aspects, the presently disclosed subject matterprovides a magnetic metal oxide nanoparticle prepared from ahigh-gravity controlled precipitation reaction, the nanoparticlecomprising: (a) iron oxide crystals having a dimension ranging fromabout 5 nm to about 100 nm; and (b) a surfactant coating; wherein thenanoparticle has a heating property of greater than about 60 W/g Fe inan alternating current (AC) magnetic field having a frequency of rangingfrom about 50 kHz and to about 1 MHz and an amplitude ranging from about0.080 kA/m to about 50 kA/m. In yet more particular aspects, thenanoparticle comprises about 76% Fe₃O₄ and about 24% γ-Fe₂O₃ and issubstantially free of Fe(OH)₂.

In yet other aspects, the presently disclosed subject matter provides abiocompatible suspension comprising a magnetic metal oxide nanoparticleprepared by a high-gravity controlled precipitation reaction and water.

In further aspects, the presently disclosed subject matter provides amethod for treating a diseased tissue, the method comprising: (a)administering to a tissue or a subject in need of treatment thereof, atherapeutically effective amount of a magnetic nanoparticle comprisingsurfactant-coated iron oxide crystals prepared from a high-gravitycontrolled precipitation process; and (b) subjecting the tissue orsubject, or a portion of the tissue or subject to an alternating current(AC) magnetic field having frequency ranging from about 50 kHz to about1 MHz and having an amplitude (peak-to-peak) ranging from about 0.080kA/m to about 50 kA/m. In particular aspects, the diseased tissuecomprises a cancer tissue.

In yet further aspects, the presently disclosed subject matter providesa magnetic nanoparticle comprising: (a) a magnetic core comprising anaggregate of at least two magnetic crystalline grains, wherein theaggregate exhibits a collective magnetic phase such that the core has anapparently single magnetic domain phase; (b) a second magnetic phase ormagnetic oxide phase differing from the collective or single domainphase of the core, wherein the second magnetic phase or magnetic oxidephase can intercalate and surround the core; wherein at least onemagnetic phase exhibits a “hard” or high-coercive behavior in a magneticfield and at least one other phase exhibits a “soft” or low-coercivebehavior in a magnetic field relative to the “hard” magnetic phase; and(c) a coating. In particular aspects, the core substantially comprisesFe₃O₄ and the second magnetic phase or magnetic oxide phasesubstantially comprises γ-Fe₂O₃.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIGS. 1A-1B show (A) a representative schematic of high gravitycontrolled precipitation (HGCP) production of particles and (B) X-raydiffraction pattern of the surface modified Fe₃O₄ particles;

FIGS. 2A-2C show (A) a scanning electron microscope (SEM) image of apresently disclosed Fe₃O₄ particle, (B) a transmission electronmicroscope (TEM) image of a presently disclosed Fe₃O₄ particle, and (C)an electron diffraction pattern of the presently disclosed Fe₃O₄particles;

FIGS. 3A-3C show (A) a representative particle size and sizedistribution measured by photon-correlation spectroscopy (PCS) of thepresently disclosed Fe₃O₄ particles, (B) a thermal gravimetric analyzer(TGA) curve of citric acid coated Fe₃O₄ particles, and (C) an IR(Infrared) spectrum of citric acid coated Fe₃O₄ particles, pure Fe₃O₄particles and pure citric acid;

FIG. 4 shows normalized heating, or specific absorption rate (SAR) plotsof citric acid coated Fe₃O₄ particles taken for three different samples.One sample was tested (square) immediately post-production, and testedagain after three months (diamond);

FIGS. 5A-5C show prostate cancer cell (PC3 and Du145) culture data (A)microscopy of particle uptake in PC3 cells; (B) cytotoxicity of PC3cells; and, (C) ICP-MS (Inductively Coupled Plasma Mass Spectrometry)data of uptake. Cellular uptake (Du-145) of citrate coated iron oxideNPs in normal and low protein medium;

FIG. 6 shows representative intratumor temperature data;

FIG. 7, panels A-C show histology data depicting (Panel A) salinecontrol with AMF (Alternating Magnetic Fields); (Panel B) particle (noAMF) control; and, (Panel C) particle+AMF with necrotic cells insideheat zone near particles and normal outside heat zone;

FIGS. 8A-B, show (FIG. 8A) Rectangular induction coil used to generateAMF for in-vitro experiments; (FIG. 8B) Magnetic field probe used tocalibrate/map the AMF amplitude;

FIG. 9 shows cells cultured with in a multi-well plate with 3 differentIONPs and a control (cells alone);

FIG. 10 shows a Measured AMF amplitude map along x- and y-axes;

FIG. 11 shows temperature rise profiles of DU145 cells treated with AMFamplitude of 20 kA/m in presence of IONPs at 3 mg of Fe/mL (arrowindicates when the AMF was turned off);

FIG. 12 shows viability of DU145 cells using neutral red assay after AMFtreatment at 20 kA/m with different IONPs;

FIG. 13 shows intratumor temperature data in a DU145 xenograft mousemodel when exposed to AMF amplitude of 29 kA/m at 155±5 kHz;

FIG. 14 shows Dynamic Light Scattering of the BNF nanoparticles in H₂O.The red line is the fit with a LogNormal distribution;

FIG. 15 shows Dynamic Light Scattering of the JHU (Johns HopkinsUniversity) nanoparticles in H₂O. The red line is the fit with aLogNormal distribution;

FIG. 16 shows Dynamic Light Scattering of the SPIO nanoparticles in H₂O.The red line is the fit with a LogNormal distribution;

FIG. 17 shows the Mossbauer Spectrum of the (dried) BNF nanoparticles at10K;

FIG. 18 shows the Mossbauer Spectrum of the (dried) JHU nanoparticles at10K;

FIG. 19 shows the Mossbauer Spectrum of the (dried) SPIO nanoparticlesat 10K;

FIGS. 20A-B, show the normalized hysteresis loop of the BNF, JHU, andSPIO nanoparticles in H₂O at 300K. (FIG. 20A) full hysteresis loop and(FIG. 20B) portion of hysteresis loop in fields achievable in thishyperthermia experiment. The moment is normalized to the ironconcentration. Sample holder and water contributions are removed, butcontributions from the dextran remain;

FIG. 21 shows the Specific Loss Power (SLP) of the BNF (red circles),JHU (green triangles), and SPIO (black squares) nanoparticles in H₂O at150 kHz as a function of peak-to-peak magnetic field amplitude. Themoment is normalized to the iron concentration;

FIG. 22 shows the nuclear scattering only contribution to the polarizedbeam SANS data of the BNF, JHU, and SPIO nanoparticles in D₂O at roomtemperature;

FIG. 23 shows the magnetic scattering contributions (parallel andperpendicular to the field) to the polarized beam SANS data of the BNFnanoparticles in D₂O at room temperature;

FIG. 24 shows the magnetic scattering contributions (parallel andperpendicular to the field) to the polarized beam SANS data of the JHUnanoparticles in D₂O at room temperature; and

FIG. 25 shows the magnetic scattering contributions (parallel andperpendicular to the field) to the polarized beam SANS data of the SPIOnanoparticles in D₂O at room temperature.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the inventions are shown. Like numbers referto like elements throughout. The presently disclosed subject matter maybe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein; rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Indeed, many modifications and other embodiments of thepresently disclosed subject matter set forth herein will come to mind toone skilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Figures. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims.

Magnetic nanoparticle hyperthermia (mNHP) is regarded as a promisingminimally invasive procedure. These nanoparticles generate heat whenexposed to alternating magnetic fields (AMFs) and thus have shown apotential for selective delivery of heat to a target such as a cancercell. Despite the great promise however, successful clinical translationhas been limited in part by technical challenges of selectivelydelivering heat only to the target tissue. Interaction of AMF withtissues also deposits heat through Joule heating via eddy currents.Considerations of patient safety thus constrain the choice of AMF powerand frequency to values that are insufficient to produce desirableheating from available nanoparticle formulations. Therefore,considerable effort must be directed to the design of particles and theAMF device to maximize the specific delivery of heat to the intendedtarget while minimizing the unintended and non-specific heating.

The presently disclosed subject matter provides iron-oxide nanoparticles(IONPs) having much higher heating capability at the clinically relevantamplitudes and frequencies than other formulations. As disclosed herein,a rectangular coil designed for treating multi well tissue culture plateis utilized and it is shown that the presently disclosed particles aresuperior to two commercially available IONPs for hyperthermia of DU145prostate cancer cells in culture. Results of pilot in-vivo experimentsusing the DU145 human prostate xenograft model in nude male mouse arereported. AMF treatment yielded an intratumor temperature rise >10° C.in <10 min heating (AMF amplitude 29 kA/m@160 kHz) with approximately 4mg nanoparticle/g tumor while maintaining rectal (core) temperature wellwithin physiological range.

I. Methods for Making Iron Oxide Nanoparticle Preparations

In some embodiments, the presently disclosed subject matter providesmethods using high gravity controlled precipitation (HGCP) technologyfor making the presently disclosed iron oxide nanoparticles.

In some embodiments, the presently disclosed subject matter provides aprocess for preparing one or more surfactant-coated magnetic metal oxideparticles, the process comprising: (a) providing a salt solution of ametal; (b) contacting the salt solution of the metal with a precipitantsolution to form a reactant solution; (c) rapidly micro-mixing thereactant solution to initiate formation of metal oxide crystals undercontrolled nucleation conditions; (d) continuing to rapidly micro-mixthe reactant solution under high gravity conditions to control crystalgrowth of one or more metal oxide particles formed therein; (e) coatingthe one or more metal oxide particles with a surfactant; (f) separatingthe one or more coated metal oxide particles from the reactant solutionand one or more by-products, if present, formed therein; and (g)exposing the one or more coated metal oxide particles to hightemperature and high pressure in an inert gas environment for a periodof time to form one or more surfactant-coated magnetic metal oxideparticles.

In some embodiments, the reactant solution comprises an iron precursorsolution comprising anhydrous FeCl₃ and FeCl₂.4H₂O in hydrochloric acid.In some embodiments, the salt solution comprises a metal salt comprisinga metal selected from the group consisting of Fe, Co, Ni, and Sm. Infurther embodiments, the metal salt comprises an anionic speciesselected from the group consisting of chloride, bromide, fluoride,iodide, nitrate (NO₃), sulfate (SO₄), chlorate (ClO₄), and phosphate(PO₄).

In some embodiments, the precipitant solution comprises ammonia. Inother embodiments, the precipitant solution comprises at least onemember selected from the group consisting of NaOH, ammonium hydroxide(NH₄OH), and another hydroxide of Group I or II elements from thePeriodic Table of elements. In further embodiments, the reactantsolution comprises at least one member selected from the groupconsisting of a hydroxide, a carbonate, and a phosphate.

In some embodiments, the coating comprises citric acid. In otherembodiments, the surfactant is selected from the group consisting of anorganic acid, a lipid, a phospholipid, an oleate, an ester, a sulfate, adiol, and a polymer. In particular embodiments, the exposing of the oneor more coated metal oxide particles to high temperature and highpressure is conducted at about 130° C. for about 5 hours.

In further embodiments, as described in more detail herein below, thepresently disclose subject matter provides one or more surfactant-coatedmagnetic metal oxide particles prepared by the presently disclosedmethods.

One characteristic of the nano- or micro-particles produced by thesemethods is that they need to provide uniform heating at many sites. Suchuniform heating requires a predictable or uniform dose and dosimetry.The alternating magnetic field (AMF) amplitude must be uniformly appliedto a large volume of tissue. The appreciable tissue volume exposurelimits field amplitude to about 15-24 kA/m. Therefore, the presentlydisclosed particles are capable of producing substantial heating at lowamplitude fields. To provide these characteristics, the presentlydisclosed subject matter provides high-gravity controlled precipitationmethods to prepare the base iron oxide crystal. The iron oxide crystalsare coated with a weak, organic acid, such as citric acid, to ensurecharge stabilization, resulting in colloid stability.

Nano- or micro-particles can be obtained by rapid micro-mixing ofreactants to enhance nucleation while suppressing crystal growth.Thorough micro-mixing leads to uniform crystal growth and thereforeuniform particle size can be obtained. On the other hand, insufficientmicro-mixing will lead to growth disparity among different nuclei,resulting in a wide particle size distribution (PSD). There are twocharacteristic time parameters in crystallization: the induction time(T) and the micro-mixing time (t_(m)). When t_(m)<<T, the nucleationrate will be nearly uniform spatially, and the PSD can be controlled ata uniform level. This can be achieved by a High Gravity ControlledPrecipitation (HGCP) reactor which utilizes a rotating packed bed tointensify mass and heat transfer in multiphase systems. During rotation,the fluids going through the packed bed are spread and split into thinfilms, threads and very fine droplets under the high shear force createdby the high gravity. This results in intense micro-mixing between thefluid elements by one to three orders of magnitude. The micro-mixingtime (t_(m)) in this process is estimated to be around the magnitude ofthe order of 10-100 μs in the presently disclosed methods.

II. Iron Oxide Nanoparticle Compositions

As used herein, the terms “nanoparticle” refers to one or morestructures that have at least one dimension, e.g., a height, width,length, and/or depth, in a range from about one nanometer (nm), i.e.,1×10⁻⁹ meters, to about 999 nm, including any integer value, andfractional values thereof, including about 1, 2, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 500, 600, 700, 800, 900, 999 nm and the like.

In some embodiments, the presently disclosed subject matter provides amagnetic metal oxide nanoparticle prepared from a high-gravitycontrolled precipitation reaction, the nanoparticle comprising: (a) ironoxide crystals having a dimension ranging from about 5 nm to about 100nm; and (b) a surfactant coating; wherein the nanoparticle has a heatingproperty of greater than about 60 W/g Fe in an alternating current (AC)magnetic field having a frequency of ranging from about 50 kHz and toabout 1 MHz and an amplitude ranging from about 0.080 kA/m to about 50kA/m.

Generally, the one or more surfactant-coated magnetic metal oxideparticles have a substantially isotropic shape and have a dimensionranging from about 50 nm to about 100 nm. More particularly, theparticles comprise about 76% Fe₃O₄ and about 24% γ-Fe₂O₃ and aresubstantially free of Fe(OH)₂.

In further embodiments, the presently disclosed subject matter providesa magnetic nanoparticle comprising: (a) a magnetic core comprising anaggregate of at least two magnetic crystalline grains, wherein theaggregate exhibits a collective magnetic phase such that the core has anapparently single magnetic domain phase; (b) a second magnetic phase ormagnetic oxide phase differing from the collective or single domainphase of the core, wherein the second magnetic phase or magnetic oxidephase can intercalate and surround the core; wherein at least onemagnetic phase exhibits a “hard” or high-coercive behavior in a magneticfield and at least one other phase exhibits a “soft” or low-coercivebehavior in a magnetic field relative to the “hard” magnetic phase; and(c) a coating. More particularly, the core substantially comprises Fe₃O₄and the second magnetic phase or magnetic oxide phase substantiallycomprises γ-Fe₂O₃.

In some embodiments, the nanoparticles may comprise a coating. Thecoating may enhance the heating properties of the nanoparticles and/ormay comprise radioactive or potentially radioactive elements. Suitablematerials for the coating include synthetic and biological polymers,copolymers and polymer blends, and inorganic materials. Polymermaterials may include various combinations of polymers of acrylates,siloxanes, styrenes, acetates, akylene glycols, alkylenes, alkyleneoxides, parylenes, lactic acid, and glycolic acid. Further suitablecoating materials include a hydrogel polymer, a histidine-containingpolymer, and a combination of a hydrogel polymer and ahistidine-containing polymer.

Coating materials may also include combinations of biological materials,such as a polysaccharide, a polyaminoacid, a protein, a lipid, aglycerol, and a fatty acid. Examples of other biological materialssuitable for use herein include heparin, heparin sulfate, chondroitinsulfate, chitin, chitosan, cellulose, dextran, alginate, starch,carbohydrate, and glycosaminoglycan. Examples of proteins useful hereininclude an extracellular matrix protein, proteoglycan, glycoprotein,albumin, peptide, and gelatin. These materials may also be used incombination with any suitable synthetic polymer material.

Inorganic coating materials may include any combination of a metal, ametal alloy, and a ceramic. Examples of ceramic materials suitable foruse herein include a hydroxyapatite, silicon carbide, carboxylate,sulfonate, phosphate, ferrite, phosphonate, and oxides of Group IVelements of the Periodic Table of Elements. These materials may form acomposite coating that may also contain one or more biological orsynthetic polymers. Where the magnetic particle is formed from amagnetic material that is biocompatible, the surface of the particleitself operates as the biocompatible coating.

The coating material may also serve to facilitate transport of thenanoparticles into a cell, a process known as transfection. Such coatingmaterials, referred to as transfection agents, include vectors, prions,polyaminoacids, cationic liposomes, amphiphiles, and non-liposomallipids or any combination thereof. A suitable vector may be a plasmid, avirus, a phage, a viron, a viral coat. The nanoprobe coating may be acomposite of any combination of transfection agent with organic andinorganic materials, such that the particular combination may betailored for a particular type of a diseased material and a specificlocation within a patient's body.

In further embodiments, the presently disclosed subject matter providesa biocompatible suspension comprising a presently disclosed magneticmetal oxide nanoparticle and water.

In still further embodiments, the presently disclosed subject matterprovides a kit for preventing and/or treating a cell disorder ordiseased tissue by using at least one magnetic metal oxide particle ofthe presently disclosed subject matter. In an embodiment, the presentlydisclosed subject matter provides a kit for treating a diseased tissue,the kit comprising a magnetic metal oxide nanoparticle prepared from ahigh-gravity controlled precipitation reaction.

III. Methods for Using Iron Oxide Nanoparticles

Metastatic cancer is characterized by diffuse disease with occult andwidespread metastatic lesions, and is typically refractory to standardof care therapies. Heat is a potent sensitizer of cancer to bothradiation and some chemotherapeutic agents. Delivering the heatselectively to cancer tumors, however, particularly those typical ofmetastatic disease represents a challenge that has not yet beenadequately addressed. Magnetic nanoparticles that are capable oflocalizing to these sites and heating when exposed to an AC magneticfield allow depositing of heat to these tumor sites with little adversedamage to surrounding normal tissue. To be effective, the nanoparticlesmust be capable of generating substantial heat (>100 W/g Fe) whenexposed to low frequency (100 kHz to 300 kHz) and low power(peak-to-peak amplitude 10 kA/m to 30 kA/m) AC fields. These latterconstraints are necessary to avoid overheating the patient bynonspecific heating that results from interactions of large volumes oftissue with the electromagnetic field.

Generally, in some embodiments, the presently disclosed subject matterprovides a method for treating a diseased tissue, the method comprising:(a) administering to a tissue or a subject in need of treatment thereof,a therapeutically effective amount of a magnetic nanoparticle comprisingsurfactant-coated iron oxide crystals prepared from a high-gravitycontrolled precipitation process; and (b) subjecting the tissue orsubject, or a portion of the tissue or subject to an alternating current(AC) magnetic field having frequency ranging from about 50 kHz to about1 MHz and having an amplitude (peak-to-peak) ranging from about 0.080kA/m to about 50 kA/m.

In one embodiment, the presently disclosed nanoparticles are used astherapeutic drugs for cell disorders. In some embodiments, the celldisorder may be, but is not limited to, cancer. In other embodiments,the presently disclosed nanoparticles may be used in other diseases,where eliminating aberrant cells or modulating an aberrant cellularfunction would be useful. Aberrant cells include, but are not limitedto, cells infected by a virus and cells infected by a bacterium.Therefore, the cell disorder may be associated with diseases, such ascancer, diseases of the immune system, pathogen-borne diseases, andundesirable targets, such as toxins, reactions to organ transplants,hormone-related diseases, and non-cancerous diseased cells or tissue.

In some embodiments, the presently disclosed subject matter has use intreating a cell disorder, such as cancer, and thus provides a method oftreating a cell disorder. More specifically, in some embodiments, themethod has use in treating or preventing a cell disorder in a subject.

The methods of the invention generally comprise contacting at least onecell with at least one nanoparticle. The methods thus can be practicedin vitro, in vivo, and ex vivo. They accordingly may be practiced, forexample, as a research method to identify compounds or to determine theeffects of compounds and concentrations of compounds, as a therapeuticmethod of treating a cell disorder, and as a method to prevent a celldisorder. In embodiments where the method is a method of treating, itcan be a method of therapy (e.g., a therapeutic method) in which theamount administered is an amount that is effective for reducing oreliminating a cell disorder. In embodiments where the method is a methodof prevention, the amount is an amount sufficient to prevent the celldisorder from occurring or sufficient to reduce the severity of the celldisorder if it does occur.

A presently disclosed nanoparticle can be targeted to a cell with adisorder by using ligands on the nanoparticle. The ligand may be apolyclonal antibody, a monoclonal antibody, a chimeric antibody, ahumanized antibody, a human antibody, a recombinant antibody, abispecific antibody, an antibody fragment, a recombinant single chainantibody fragment, or any combination of the above.

The choice of a marker (antigen) may be important in the targetedtherapy methods of the presently disclosed subject matter. Although notlimited thereto, use and selection of markers is most prevalent incancer immunotherapy. For breast cancer and its metastases, a specificmarker or markers may be selected from cell surface markers such as, forexample, members of the MUC-type mucin family, an epithelial growthfactor (EGFR) receptor, a carcinoembryonic antigen (CEA), a humancarcinoma antigen, a vascular endothelial growth factor (VEGF) antigen,a melanoma antigen (MAGE) gene, family antigen, a T/Tn antigen, ahormone receptor, growth factor receptors, a clusterdesignation/differentiation (CD) antigen, a tumor suppressor gene, acell cycle regulator, an oncogene, an oncogene receptor, a proliferationmarker, an adhesion molecule, a proteinase involved in degradation ofextracellular matrix, a malignant transformation related factor, anapoptosis related factor, a human carcinoma antigen, glycoproteinantigens, DF3, 4F2, MGFM antigens, breast tumor antigen CA 15-3,calponin, cathepsin, CD 31 antigen, proliferating cell nuclear antigen10 (PC 10), and pS2.

For other forms of cancer and their metastases, a specific marker ormarkers may be selected from cell surface markers such as, for example,a member of vascular endothelial growth factor receptor (VEGFR) family,a member of carcinoembryonic antigen (CEA) family, a type ofanti-idiotypic mAB, a type of ganglioside mimic, a member of clusterdesignation/differentiation antigens, a member of epidermal growthfactor receptor (EGFR) family, a type of a cellular adhesion molecule, amember of MUC-type mucin family, a type of cancer antigen (CA), a typeof a matrix metalloproteinase, a type of glycoprotein antigen, a type ofmelanoma associated antigen (MAA), a proteolytic enzyme, a calmodulin, amember of tumor necrosis factor (TNF) receptor family, a type ofangiogenesis marker, a melanoma antigen recognized by T cells (MART)antigen, a member of melanoma antigen encoding gene (MAGE) family, aprostate membrane specific antigen (PMSA), a small cell lung carcinomaantigen (SCLCA), a T/Tn antigen, a hormone receptor, a tumor suppressorgene antigen, a cell cycle regulator antigen, an oncogene antigen, anoncogene receptor antigen, a proliferation marker, a proteinase involvedin degradation of extracellular matrix, a malignant transformationrelated factor, an apoptosis-related factor, a type of human carcinomaantigen.

For ovarian cancers and their metastases, a specific marker or markersmay be selected from cell surface markers such as, for example, one ofERBB2 (HER-2) antigen and CD64 antigen. For ovarian and/or gastriccancers and their metastases, a specific marker or markers may beselected from cell surface markers such as, for example, a polymorphicepithelial mucin (PEM). For ovarian cancers and their metastases, aspecific marker or markers may be selected from cell surface markerssuch as, for example, one of cancer antigen 125 (CA125) or matrixmetalloproteinase 2 (MMP-2). For gastric cancers and their metastases, aspecific marker or markers may be selected from cell surface markerssuch as, for example, one of CA 19-9 antigen and CA242 antigen.

For non-small cell lung cancer (NSCLC), colorectal cancer (CRC) andtheir metastases, a specific marker or markers may be selected from cellsurface markers such as, for example, vascular endothelial growth factorreceptor (VEGFR), anti-idiotypic mAb, and carcinoembryonic antigen (CEA)mimic. For at least one of small-cell lung cancer (SCLC), malignantmelanoma, and their metastases, a specific marker or markers may beselected from cell surface markers such as, for example, anti-idiotypicmAB or GD3 ganglioside mimic. For melanoma cancers and their metastases,a specific marker or markers may be selected from cell surface markerssuch as, for example, a melanoma associated antigen (MAA). For smallcell lung cancers and their metastases, a specific marker or markers maybe selected from cell surface markers such as, for example, a small celllung carcinoma antigen (SCLCA).

For colorectal cancer (CRC) and/or locally advanced or metastatic headand/or neck cancer, a specific marker or markers may be selected fromcell surface markers such as, for example, epidermal growth factorreceptor (EGFR). For Duke's colorectal cancer (CRC) and its metastases,a specific marker or markers may be selected from cell surface markerssuch as, for example, Ep-CAM antigen.

For non-Hodgkin's lymphoma (NHL) and its metastases, a specific markeror markers may be selected from cell surface markers such as, forexample, cluster designation/differentiation (CD) 20 antigen or CD22antigen. For B-cell chronic lymphocytic leukemia and associatedmetastases, a specific marker or markers may be selected from cellsurface markers such as, for example, CD52 antigen. For acutemyelogenous leukemia and its metastases, a specific marker or markersmay be selected from cell surface markers such as, for example, CD33antigen.

For prostate cancers and their metastases, a specific marker or markersmay be selected from cell surface markers such as, for example, prostatemembrane specific antigen (PMSA). For carcinomatous meningitis and theirmetastases, a specific marker or markers may be selected from cellsurface markers such as, for example, one of a vascular endothelialgrowth factor receptor (VEGFR) or an epithelial associated glycoprotein,for example, HMFGI (human milk fat globulin) antigen.

For lung, ovarian, colon, and melanoma cancers and their metastases, aspecific marker or markers may be selected from cell surface markerssuch as, for example, B7-H1 protein. For colon, breast, lung, stomach,cervix, and uterine cancers and their metastases, a specific marker ormarkers may be selected from cell surface markers such as, for example,TRAIL Receptor-1 protein, a member of the tumor necrosis factor receptorfamily of proteins. For ovarian, pancreatic, non-small cell lung,breast, and head and neck cancers and their metastases, a specificmarker or markers may be selected from cell surface markers such as, forexample, EGFR (epidermal growth factor receptor).

For anti-angiogenesis targeting of tumor blood supply, a specific markeror markers may be selected from cell surface markers such as, forexample, Integrin αvβ3, a cell surface marker specific to endothelialcells of growing blood vessels.

For targeting of colon and bladder cancer and their metastases, aspecific marker or markers may be selected from cell surface markerssuch as, for example, RAS, a signaling molecule that transmits signalsfrom the external environment to the nucleus. A mutated form of RAS isfound in many cancers.

The cell comprising the target may express several types of markers. Oneor more nanoparticles may attach to the cell via a ligand. Thenanoparticle may be designed such it remains externally on the cell ormay be internalized into the cell comprising the target. Once bound tothe cell, the magnetic nanoparticle heats in response to the energyabsorbed. For example, the magnetic nanoparticle may heat throughhysteresis losses in response to an AMF. The heat may pass through thecoating or through interstitial regions to the cell, for example viaconvection, conduction, radiation, or any combination of these heattransfer mechanisms. The heated cell becomes damaged, preferably in amanner that causes irreparable damage. When a sufficient amount ofenergy is transferred by the nanoparticle to the cell, the cell dies vianecrosis, apoptosis or another mechanism.

The nanoparticles may comprise one or more ligands that target andattach to a biological marker. Suitable ligands for use herein include,but are not limited to, proteins, peptides, antibodies, antibodyfragments, saccharides, carbohydrates, glycans, cytokines, chemokines,nucleotides, lectins, lipids, receptors, steroids, neurotransmitters,Cluster Designation/Differentiation (CD) markers, and imprinted polymersand the like. The preferred protein ligands include, for example, cellsurface proteins, membrane proteins, proteoglycans, glycoproteins,peptides and the like. The preferred nucleotide ligands include, forexample, complete nucleotides, complimentary nucleotides, and nucleotidefragments. The preferred lipid ligands include, for example,phospholipids, glycolipids, and the like.

Covalent bonding may be achieved with a linker molecule. Examples offunctional groups used in linking reactions include amines, sulfhydryls,carbohydrates, carboxyls, hydroxyls and the like. The linking agent maybe a homobifunctional or heterobifunctional crosslinking reagent, forexample, carbodiimides, sulfo-NHS esters linkers and the like. Thelinking agent may also be an aldehyde crosslinking reagent such asglutaraldehyde.

In an embodiment, the ligand may target one or more markers on a cancercell. In another embodiment, the ligand may target a predeterminedtarget associated with a disease of the patient's immune system. Theparticular target and one or more ligands may be specific to, but notlimited to, the type of the immune disease. The ligand may have anaffinity for a cell marker or markers of interest. The marker or markersmay be selected such that they represent a viable target on T cells or Bcells of the patient's immune system. The ligand may have an affinityfor a target associated with a disease of the patient's immune systemsuch as, for example, a protein, a cytokine, a chemokine, an infectiousorganism, and the like. For rheumatoid arthritis, a specific marker ormarkers may be selected from cell surface markers, such as, for example,one of CD52 antigen, tumor necrosis factor (TNF), and CD25 antigen. Forrheumatoid arthritis and/or vasculitis, a specific marker or markers maybe selected from cell surface markers such as, for example, CD4 antigen.For vasculitis, a specific marker or markers may be selected from cellsurface markers such as, for example, CD18 antigen. For multiplesclerosis, a specific marker or markers may be selected from cellsurface markers such as, for example, CD52 antigen.

In still another embodiment, the ligand targets a predetermined targetassociated with a pathogen-borne condition. The particular target andligand may be specific to, but not limited to, the type of thepathogen-borne condition. A pathogen is defined as any disease-producingagent such as, for example, a bacterium, a virus, a microorganism, afungus, and a parasite. For a pathogen-borne condition, the ligand fortherapy utilizing nanoparticles may be selected to target the pathogenitself. For a bacterial condition, a predetermined target may be thebacteria itself, for example, one of Escherichia coli or Bacillusanthracis. For a viral condition, a predetermined target may be thevirus itself, for example, one of Cytomegalovirus (CMV), Epstein-Barrvirus (EBV), a hepatitis virus, such as Hepatitis B virus, humanimmunodeficiency virus, such as HIV, HIV-1, or HIV-2, or a herpes virus,such as Herpes virus 6. For a parasitic condition, a predeterminedtarget may be the parasite itself, for example, one of Trypanasomacruzi, Kinetoplastid, Schistosoma mansoni, Schistosoma japonicum orSchistosoma brucei. For a fungal condition, a predetermined target maybe the fungus itself, for example, one of Aspergillus, Cryptococcusneoformans or Rhizomucor.

In another embodiment, the ligand targets a predetermined targetassociated with an undesirable target material. The particular targetand ligand may be specific to, but not limited to, the type of theundesirable target. An undesirable target is a target that may be anundesirable material. Undesirable material is material associated with adisease or an undesirable condition, but which may also be present in anormal condition. For example, the undesirable material may be presentat elevated concentrations or otherwise be altered in the disease orundesirable state. The ligand may have an affinity for the undesirabletarget or for biological molecular pathways related to the undesirabletarget. The ligand may have an affinity for a cell marker or markersassociated with the undesirable target material. For arteriosclerosis, apredetermined target may be, for example, apolipoprotein B on lowdensity lipoprotein (LDL). An undesirable material may be adipose tissueor cellulite for obesity, associated with obesity, or a precursor toobesity. A predetermined marker or markers for obesity maybe selectedfrom cell surface markers such as, for example, one of gastricinhibitory polypeptide receptor and CD36 antigen. Another undesirablepredetermined target may be clotted blood.

In another embodiment, the ligand targets a predetermined targetassociated with a reaction to an organ transplanted into the patient.The particular target and ligand may be specific to, but not limited to,the type of organ transplant. The ligand may have an affinity for abiological molecule associated with a reaction to an organ transplant.The ligand may have an affinity for a cell marker or markers associatedwith a reaction to an organ transplant. The marker or markers may beselected such that they represent a viable target on T cells or B cellsof the patient's immune system.

In another embodiment, the ligand targets a predetermined targetassociated with a toxin in the patient. A toxin is defined as any poisonproduced by an organism including, but not limited to, bacterial toxins,plant toxins, insect toxin, animal toxins, and man-made toxins. Theparticular target and ligand may be specific to, but not limited to, thetype of toxin. The ligand may have an affinity for the toxin or abiological molecule associated with a reaction to the toxin. The ligandmay have an affinity for a cell marker or markers associated with areaction to the toxin. A bacterial toxin target may be, for example, oneof Cholera toxin, Diphtheria toxin, and Clostridium botulinus toxin. Aninsect toxin may be, for example, bee venom. An animal toxin may be, forexample, snake toxin, for example, Crotalus durissus terrificus venom.

In another embodiment, the ligand targets a predetermined targetassociated with a hormone-related disease. The particular target andligand may be specific to, but not limited to, a particular hormonedisease. The ligand may have an affinity for a hormone or a biologicalmolecule associated with the hormone pathway. The ligand may have anaffinity for a cell marker or markers associated with the hormonedisease. For estrogen-related disease or conditions, a predeterminedtarget may be, for example, estrogen or cell surface marker or markerssuch as, for example, estrogen receptor. For human growth hormonedisease, the predetermined target may be, for example, human growthhormone.

In another embodiment, the ligand targets a predetermined targetassociated with non-cancerous disease material. The particular targetand ligand may be specific to, but not limited to, a particularnon-cancerous disease material. The ligand may have an affinity for abiological molecule associated with the non-cancerous disease material.The ligand may have an affinity for a cell marker or markers associatedwith the non-cancerous disease material. For Alzheimer's disease, apredetermined target may be, for example, amyloid B protein and itsdeposits, or apolipoprotein and its deposits.

In another embodiment, the ligand targets a proteinaceous pathogen. Asan example, for prion diseases also known as transmissible spongiformencephalopathies, a predetermined target may be, for example, Prionprotein 3F4.

In an embodiment, the nanoparticle is targeted to a cancer cell. Inanother embodiment, the particles will localize to a tumor, such as ametastatic tumor or micrometastases. Types of cancers include, but arenot limited to, bladder, lung, breast, melanoma, colon, rectal,non-Hodgkin lymphoma, endometrial, pancreatic, kidney, prostate,leukemia, thyroid, and the like.

IV. Definitions

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

By “disease” or “cell disorder”, it is meant any condition, dysfunction,or disorder that damages or interferes with the normal function of acell, tissue, or organ.

The term “AMF” (an abbreviation for alternating magnetic field), as usedherein, refers to a magnetic field that changes the direction of itsfield vector periodically, typically in a sinusoidal, triangular,rectangular or similar shape pattern, with a frequency of in the rangeof from about 80 kHz to about 800 kHz. The AMF may also be added to astatic magnetic field, such that only the AMF component of the resultingmagnetic field vector changes direction. It will be appreciated that analternating magnetic field is accompanied by an alternating electricfield and is electromagnetic in nature.

The term “coating”, as used herein, refers to a material, combination ofmaterials, or covering of the magnetic nanoparticle, comprising asuitable biocompatible material that serves to affect in vivo transportof the nanoparticle throughout the patient, and facilitates uptake andretention by diseased tissues and cell.

In some embodiments, the term “nanoparticle”, as used herein, refers toa targeted nanoparticle that may comprise a magnetic nanoparticle core,coating, linker, and targeting ligand, that is used to selectively treattissue by heating in response to an alternating magnetic field (AMF).Additionally, the nanoparticle may comprise a radioactive source orspecies that may become radioactive when exposed to an appropriateenergy source. The nanoparticle may also comprise a chemotherapeuticagent, such as doxorubicin. In some embodiments, a nanoparticlecomprises a coating, is attached to a target (such as a cell) by one ormore targeting ligands.

The term “cell disorder” or “diseased tissue”, as used herein, refers totissue or cells associated with cancer of any type, such as bone marrow,lung, vascular, neuro, colon, ovarian, breast and prostate cancer;diseases of the immune system, such as AIDS; pathogen-borne diseases,which can be bacterial, viral, parasitic, or fungal, examples ofpathogen-borne diseases include HIV, tuberculosis and malaria;hormone-related diseases, such as obesity; vascular system diseases;central nervous system diseases, such as multiple sclerosis; andundesirable matter, such as adverse angiogenesis, restenosis,amyloidosis, toxins, reaction-by-products associated with organtransplants, and other abnormal cell or tissue growth. The term“ligand”, as used herein, refers to a molecule or compound that attachesto a nanoparticle and targets and attaches to a biological marker.

The terms “linker” or “linker molecule,” as used herein, refer to anagent that targets particular functional groups on a ligand and on amagnetic particle or a coating, and thus forms a covalent link betweenany two of these.

The term “target”, as used herein, refers to the matter for whichdeactivation, rupture, disruption or destruction is desired, such as adiseased cell, a pathogen, or other undesirable matter. A marker may beattached to the target.

By “contacting”, it is meant any action that results in at least onemolecule of one of the presently disclosed nano- or micro-particlesphysically contacting at least one cell. It thus may comprise exposingthe cell(s) to the particle in an amount sufficient to result in contactof at least one particle with at least one cell. The method can bepracticed in vitro or ex vivo, by introducing, and preferably mixing,the compound and cells in a controlled environment, such as a culturedish or tube. The method can be practiced in vivo, in which casecontacting means exposing at least one cell in a subject to at least oneparticle of the presently disclosed subject matter, such asadministering the particle to a subject via any suitable route. Themethod for administration of a magnetic material composition to asubject may include intraperitoneal injection, intravascular injection,intramuscular injection, subcutaneous injection, topical, inhalation,ingestion, rectal insertion, wash, lavage, rinse, or extracorporealadministration into a patient's bodily materials. According to thepresently disclosed subject matter, contacting may comprise introducing,exposing, and the like, the particle at a site distant to the cells tobe contacted, and allowing the bodily functions of the subject, ornatural (e.g., diffusion) or man-induced (e.g., swirling) movements offluids to result in contact of the particle and cell(s).

The subject treated by the presently disclosed methods in their manyembodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes, such as for the treatment of an existing condition ordisease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease. Thus,the terms “subject” and “patient” are used interchangeably herein.

The “effective amount” of an active agent or drug delivery device refersto the amount necessary to elicit the desired biological response. Aswill be appreciated by those of ordinary skill in this art, theeffective amount of an agent or device may vary depending on suchfactors as the desired biological endpoint, the agent to be delivered,the composition of the encapsulating matrix, the target tissue, and thelike.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 Materials and Methods for Preparation of Iron OxideNanoparticles

Anhydrous iron(III) chloride (FeCl₃) and anhydrous citric acid werepurchased from GCE laboratory chemicals. Iron(II) chloride tetrahydrate(FeCl₂.4H₂O) and ammonia solution (25%) were purchased from Uni ChemChemical and Merck Co (Whitehouse Station, N.J.), respectively. All thesolvents and reagents were of analytical grade and used without furtherpurification.

Magnetite (Fe₃O₄) particles were prepared in a small scale HGCP platformvia co-precipitation method. Iron precursor solution was freshlyprepared by 24.4 g of anhydrous FeCl₃ and 14.9 g of FeCl₂.4H₂O in 500 mLof 0.74 M hydrochloric acid and kept under inert gas protection at 90°C. Under continuous flow of nitrogen gas, excess 25% ammonia solutionwas added with vigorous stirring. The reaction mixture turned blackimmediately and 40 mL of 0.24 M citric acid solution was added. Reactionwas continued for 1 hour and magnetite particles were allowed to settle.The supernatant was decanted and settlement was isolated bycentrifugation. The particles were washed several times bysolvent/anti-solvent precipitation with water and acetone to achievedispersion at pH 6-8. The trace of acetone was removed under reducedpressure at 60° C. for 15 minutes before the dispersion were treatedhydrothermally at 130° C. for 5 hours. The final dispersion was placedunder ultrasonic to ensure well dispersion. The final products werepurged by argon gas and kept in a sealed bottle to prevent oxidation ofFe₃O₄ to Fe₂O₃.

Particle Size Analysis by DLS

The hydrodynamic diameter of the Fe₃O₄ was measured by a Horiba LB-550Dynamic Light Scattering Particle Size distribution Analyzer with 0.01wt % iron Oxide aqueous suspension. The distribution base was set tovolume mode and the Refractive Index of Fe₃O₄ and deionized water (DI)water were set at 2.42 and 1.33, respectively.

Transmission Electron Microscope (TEM) and Scanning Electron Microscope(SEM)

The TEM image was acquired on a 200 kV JEOL 2010 transmission electronmicroscope. The specimen was prepared by placing a drop of suspensioncontaining a drop of aqueous Fe₃O₄ (3 mass %) in 20 mL methanol onto acarbon coated copper grid followed by drying at room temperature for 24hours. The size and morphology of the samples were also investigatedwith a KYKY-2800B scanning electron microscope (SEM).

X-Ray Diffraction (XRD) Analysis

The crystal structure and the phase purity and size of the samples wereexamined by X-ray powder diffraction (XRD) on a Philips expertdiffractometer with Cu Kα radiation at room temperature. XRD wasperformed on powdered samples over the 2θ range of 28° to 67° with astep width of 0.1″ and a sampling time of 4 seconds.

Thermal Gravimetric Analyzer (TGA)

The amount of surfactant coating on the Fe₃O₄ particles was examined ona thermal gravimetric analyzer (TGA), TA instrument 2050. Heating rangewas 25° C. to 800° C. under a nitrogen atmosphere with a heating rate of20° C./min.

Iron Concentration Measurement by ICP-AES

The final concentration of Fe₃O₄ in the aqueous suspension wasdetermined by a dual-view Optima 5300 DV ICP-AES (Inductively CoupledPlasma Atomic Emission spectroscopy) system in the Elemental AnalysisLaboratory (EAL), Department of Chemistry, National University ofSingapore (NUS), Singapore. The sample digestion and preparation werealso performed at EAL based on the Milestone microwave laboratorysystem.

Specific Absorption Rate (SAR)

The SAR measurements were performed by the Department of RadiationOncology & Molecular Radiation Sciences at Johns Hopkins University,(Baltimore, Md.).

Example 2 Characterization of Iron Oxide Nanoparticles

A representative schematic of high gravity controlled precipitation(HGCP) production of particles is shown in FIG. 1A. X-Ray powderdiffraction (XRD) analysis confirmed the formation of Fe₃O₄ (FIG. 1B).Six characteristic peaks for Fe₃O₄ (2θ=30.3, 35.6, 43.3, 53.7, 57.3 and62.9) as reported were observed.

The as-synthesized surface modified Fe₃O₄ particles were observed by SEMand TEM (FIG. 2). Both SEM and TEM images shown in FIGS. 2A and 2B,respectively, reveal that the single particle size of these Fe₃O₄particles is around 15-20 nm and the TEM image shows that theseparticles have off-cubic morphology. The distinct ring pattern of theelectron diffraction pattern shown in FIG. 2C signifies that thesynthesized Fe₃O₄ is of high degree of crystallinity.

The surface modified Fe₃O₄ nanoparticles dispersed well in DI water andremained stable for more than 6 months with a mean hydrodynamic particlesize of around 60 nm. The representative particle size distributiondetermined using dynamic light scattering is shown in FIG. 3A.

The amount of surfactants coating on the particle was monitored by TGAwhich revealed a total weight loss of 5% as shown in FIG. 3B. There wasapproximately 1.5% weight loss in the stage of 50˜150° C. followed byanother 3.5% loss in the stage of 200˜350° C. and remained almostconstant after 400° C. The first loss was caused by dehydration of thesurface moisture while the latter was caused by the decomposition of thecoated biocompatible acid. The Fe₃O₄ particles contained around 95%magnetite on a mass basis.

IR spectra were recorded on an IR Prestige-21 spectrometer (SHIMADZU,Columbia, Md.; 400-4000 cm⁻¹ KBr pellets). The IR spectra of citric acidcoated Fe₃O₄, pure Fe₃O₄, and pure citric acid are shown as curves a, band c in FIG. 3C. Comparing the curves, the appearance of thecharacteristic absorption peaks at 590 cm⁻¹ in curve a, which indicatesthe presence of Fe₃O₄. While the absorption of free citric acid ataround 1700⁻¹ to 750 cm⁻¹ (C═O) is not found in curve a, the absorptionsat 1589.9 and 1396.9 cm⁻¹ are assigned as the stretching vibration ofCOO group of the citrate coated Fe₃O₄.

Three batches of citric acid coated Fe₃O₄ with mean size around 60 nmwere prepared as described. The SAR (Specific Absorption Rate) plotsshowed that these particles were around 114 W/g Fe at magnetic field andfrequency at 253 Oersteds and 150 kHz, respectively. The SAR plots inFIG. 4 demonstrate the reproducibility of the synthesis of the Fe₃O₄particles with consistent SAR performance. In addition, no significantchanges were observed in the SAR performance when the SAR of one ofthese samples was retested after 2 months.

The prepared nanoparticles were internalized by human prostate cancercells (PC3 and Du145) for demonstration (FIG. 5A). Exposure to particlesdid not produce significant cytotoxicity, except for high concentrationsof particle exposure (FIG. 5B). Exposure of Du145 cancer cells tocitrate-coated particles in both low and high serum media (bovine serumalbumin, BSA) demonstrated slightly increased tendency to internalizeparticles when cells were conditioned by low serum media, suggestingincreased cell-particle interactions. Protein in media are hypothesizedto form a “corona” on the panicles upon contact with media that caninfluence cell-particle interactions. Thus, particle coating forbiocompatibility and blood circulation following intravenous deliverymay be an important consideration for systemic delivery of moleculartargeted nanoparticles for non-invasive therapy.

Nanoparticies directly injected into Du145 (human prostate) subcutaneousthigh xenografts in mice will produce substantial heat within the tumorwhen exposed to AMF. By contrast, the measured rectal temperature of thesame mouse (a surrogate measure of body core temperature) duringtreatment does not rise substantially, demonstrating the effective andlocalized deposition of heat by nanoparticles (FIG. 6). Histologicanalysis from sectioned and excised tumors shows viable cells withinjection of saline (phosphate buffered saline, no nanoparticles) andAMF (FIG. 7A). Also, tumor cells are viable when injected withnanoparticle formulation and no AMF (FIG. 7B). When both nanoparticlesand AMF are combined in the tumor following intratumor delivery,widespread necrosis (cell death) is observed in the vicinity of thenanoparticles (FIG. 7C). Cell death is localized to nanoparticledistribution, as demonstrated by cell viability for regions of tumor notcontaining nanoparticles as demonstrated by intact nuclei and cellmembranes in FIG. 7C.

Fe₃O₄ nanoparticles aqueous dispersion with mean hydrodynamic particlesize of around 60 nm and SAR of around 114 W/g Fe at magnetic field andfrequency at 253 Oersteds and 150 kHz, respectively were preparedsuccessfully.

Example 3 Materials and Methods for In Vitro and In Vivo ExperimentsRectangular Induction Coil for Cell Culture Experiments

The design of rectangular induction coil used in the study is describedelsewhere (Nemkov et al., 2011). Briefly, the inductor consists of twosingle turn (square) copper tubes connected in parallel. At the ends ofthe copper tubes, copper blocks are connected to prevent the magneticfield from diverging at the ends of the coil. Fluxtrol 75 (FluxtrolInc., Auburn Hills, Mich.) is used as a magnetic core and is placed inthe area inside of the tubes and over the upper edge of the tube. Awater-cooled copper plate is placed on the outside of the cores toextract heat from the magnetic core generated during operation. Apicture of the induction coil developed for heating the multi-wellplates is shown in FIG. 8.

Field Mapping

The distribution of the magnetic field was measured using a magneticfield probe (AMF Life Systems, LLC, Rochester, Mich.), shown in FIG. 8B,and methods previously described (Bordelon et al., 2012). Magnetic fieldmeasurements were taken at the central height between the turns.Measurements were taken at multiple locations along the centerline. Fivemeasurements were made in the coil length at 3.5, 7, 10.5, 14 and 17.5cm from the copper plates at the start of the coil head. In the coilwidth, fifteen measurements were taken. One measurement every cm leftand right from the centerline for 7 cm. The measurements were repeated 4times and the results were averaged.

Thermal Management

Several thermal management methods were utilized to maintain a safe coiloperating temperature and to shield cell culture samples fromenvironmental temperatures in excess of 39° C. Nemkov et al., 2011 havedescribed the design criteria and approaches for thermal management.Briefly, heat extraction through water cooling, concentrator materialselection/orientation for reduced losses and better heat transfer,reduction of magnetic flux density by design modification, selection ofproper materials for adhesion, and intermediate layers were utilized.Thermal load management during AMF system operation required for therectangular coil h described previously (Nemkov et al., 2011). Initialtesting of the coil was performed at a power supply voltage of 500 V,which led to an inductor voltage of 480 V. At this voltage the meanfield strength along the center was 22.6 kA/m at 155 kHz. Proportionallyfor 31.8 kA/m at 150 kHz, 654 V will be needed in the coil head, a valueclose to the 650 V predicted by simulation. Thermal measurements weretaken of the coil as well. The hottest surfaces of the inductor were inthe same areas as predicted by simulation (Nemkov et al., 2011). Theeffectiveness of the cooling plates/recirculator system was tested bymonitoring the temperature on the surface of the bottom cooling plateusing fiber optic probes (FISO Technologies, Quebec, Canada).Measurement of the temperature on the bottom plate is most relevant asit is in the closest contact with the sample.

Iron-Oxide Nanoparticles (IONPs)

Two commercially available IONPs were chosen to compare in-vitro heatingcharacteristics with the newly developed particles.

Bionized NanoFerrite (BNF) and Nanomag-D-Spio Particles

Suspensions of starch-coated magnetite (Fe³O₄) core-shell particlesBNF-Starch (catalog no. 10-00-102) and nanomag-D-spio (catalog no.79-00-102) were obtained from micromod Partikeltechnologie, GmbH(Rostock, Germany). Synthesis procedure and structural, heating andmagnetic properties of these particles have been previously described(Gruettner et al., 2007; Gruttner et al., 1997; Dennis et al., 2009).Particle size of approximately 100 nm and AMF amplitude dependentheating characteristics are reported elsewhere (Bordelon et al., 2011).

JHU-MION (Newly Developed Particles)

Suspension of dextran-coated JHU iron oxide core-shell particles wereprepared by Micromod Partikeltechnologie, GmbH (Rostock, Germany). Theiron oxide core was prepared separately using a proprietary high-gravitycontrolled precipitation (HGCP) method (Nanomaterials Technologies,Ltd., Singapore) from aqueous solutions of precursor FeCl₂ and NH₄OH.Detailed description of synthesis and particle structure and magneticcharacterization are in preparation for publication. The JHU-MION ironoxide cores were dextran coated using methods similar to those describedfor BNF particles (Gruettner et al., 2007) and was used as received.Size (˜100 nm) and stability of the dextran-JHU-MION particles weretested. FIG. 2A shows a scanning electron microscope (SEM) image of theJHU-MIONs.

In Vitro Experiments

About 10,000 DU145 prostate cancer cells were added per well of two48-well culture plates and allowed to grow in RPMI1640 without phenolred media (Invitrogen, Carlsbad, Calif.) containing 10% fetal bovineserum for 4 days at 37° C. and 5% CO2. Just prior to AMF treatment theindicated iron oxide nanoparticles were added in triplicates to thefinal concentrations of: 0.5, 1, 2, and 3 mg/mL Fe per well. 12 wellscontained only cells and media to serve as control (FIG. 9). One of thetwo identical plates was subjected to AMF treatment in the coildescribed above. The second culture plate was used to characterize thecytotoxity from the particles alone.

Temperature Measurements

To monitor the change in temperature, 4 FISO RF-resistant fiber optictemperature probes (FISO, Inc., Quebec, Canada) were taped to the bottomof the plate under the wells containing the highest iron concentration(one for each particle and one for the control). In short, the coilgeometry/dimensions make it impossible to fully insert an optical fibertemperature probe into the media without breaking the probe. To overcomethis, “surrogate” temperatures were measured at the bottom surface ofthe plate under representative wells. This method or configurationprovides reasonably accurate relative measure of temperature and heatingfor comparisons among the wells. It, however fails to provide an“absolute” measure of the temperature in the system. It is assumed thatthe temperature in all wells (before AMF heating, and in no particlecontrols) begins at the same value which is determined by thetemperature set point (37° C.) of the water-jacket thermal managementsystem. For each experiment, the culture plate was allowed toequilibrate with the coil for ˜15 min prior to application of AMF. AMFwas applied at 500V for 30 minutes.

Viability Assay

After the treatment, the plates were washed twice with the same growthmedia and kept at 37° C. and 5% CO₂ for additional 4 days. The viabilitywas assessed by neutral red uptake assay as described (Repetto et al.,2008). The neutral red assay is based on the ability of viable cells toincorporate and bind the neutral red dye which is slightly cationic.Neutral red is retained in the lysosomes of live and undamaged cells andcan be extracted by a neutral destaining solution (49% ethanol, 1%glacial acetic acid in deionized water; Repetto et al., 2008). Briefly,200 μL of cleared 40 μg/mL solution of neutral red (Sigma-Aldrich N4638,St. Louis, Mo.) was added to each well. After incubation at 37° C. for 3hours, the neutral red medium was removed and the wells were washed withphosphate buffered saline. Neutral red destain solution was added (300μL per well) and the plate was then placed on a shaker for about 20 min.Optical density (@450 nm) was measured by microplate reader spectrometer(SpectraMax M5, Molecular Devices, Sunnyvale, Calif.).

Animal Model

One 5-7 week old male nude mouse (Hsd: Athymic Nude-Foxn1^(nu), HarlanLabs, Indianapolis, Ind.) weighing ˜20 g prior to the treatment was usedin this study. Male nude mice were selected for their relevance to ourongoing studies on prostate cancer therapy. The experiments wereconducted using protocols approved by the Johns Hopkins InstitutionalAnimal Care and Use Committee (Baltimore, Md.). Xenograft tumors wereobtained by injecting 1×10⁶ DU145 cells subcutaneously in the flank ofmale athymic nude mouse. Once the tumor reached to the size of about0.15±0.02 cm³, IONPs were directly injected into the tumor.

Alternating Magnetic Field (AMF) System

In some embodiments, a high-throughput device capable of accommodatingmulti-well dishes is provided. Further, such a device must providetemperature control that is suitable for cell culture experiments(within physiologic limits) with continuous duty operation for more thanone hour. This will ensure that the only heat stress on the cellsoriginates from the nanoparticles and not from thermal losses of thedevice. The inductor needs to sustain low temperatures not only for thereliability of data but also to prevent degradation of the inductor fora long life cycle.

To address these requirements, a rectangular, modified Helmholtz coilthat produces a homogeneous flux density AMF (<10% variation) throughouta volume having dimensions 86 mm×127 mm×19 mm with area sufficient toaccommodate larger culture dishes (80 mm×120 mm), including a standard96-well plate, was developed. The coil developed for this purpose hasbeen previously described and characterized (Nemkov et al., 2011).

More particularly, the AMF system used for the in-vivo experiments hasbeen described elsewhere (Kumar et al., 2013). A brief description isprovided here. The AMF system comprises three main components: (a) thepower source; (b) an external impedance matching (capacitance) network;and, (c) the load. The load comprises an inductor, or solenoid coil.

Results obtained using this coil with cell cultures containing multiplenanoparticle formulations for comparison are provided herein. Resultsobtained from hyperthermia experiments using the new IONP (JHU-MIONs)formulation in both cell culture (with modified Helmholtz coil) andmouse experiments are presented. The JHU-MIONs have higher heatingcapability at low H-values (<30 KA/m) compared to commercially availablebionized-nanoferrite (BNFs).

The power source, matching network, and inductor were cooled with anindustrial (80 kW rated) closed-loop circulating water/water coolingsystem comprising a 200-L reservoir of distilled water that is pumpedthrough the RF system at a flow rate of 170 L/min and pressure 6 atm.The power supply was an 80-kW induction heating system manufactured byPPECO (Watsonville, Calif.) that provides an alternating current to aresonant circuit with variable frequency between 135 kHz and 440 kHz.The power supply (source) impedance was adjusted to match the coil andcapacitance network by adjusting its internal inductance andcapacitance. The external capacitance network (AMF Life Systems, Inc.,Auburn Hills, Mich.) was adjusted for stable oscillation at 160±1 kHz.

Inductor or Solenoid Coil

For mNHP research, simple solenoids are typically used to performexperiments with small animal models and cancer cells in culture. Whilethe solenoid geometry is adequate for small animal experiments, giventhe cylindrical shape of both coil and animal subject; this geometry isproblematic for cell culture experiments. Few culture dishes areavailable that accommodate both necessary numbers of cells (>10,000)with a diameter sufficiently small to fit within the typical solenoidemployed which often have a diameter less than 3 cm. Further, simplesolenoid coils generate inhomogeneous fields necessitating precisesample positioning. To surmount these challenges, cell cultureexperiments are often performed by suspending the cells in a pelletform. While this is technically feasible, such experimental designsrequire additional manipulation of cells, and treatment in a state ofsuspension. These manipulations limit both the number of experimentalsamples that can be treated simultaneously and require many controls(Hedayati et al., 2013).

A four-turn solenoid with inner diameter of 45.5 mm, outer diameter of57.5 mm, and a length of 32 mm was constructed from dehydrated annealedsoft-copper refrigerator tubing having 6.4 mm outer diameter (OD).Measurements of the AMF amplitude were taken in the center of the coilwith a magnetic field probe (AMF Life Systems, Inc., Auburn Hills,Mich.). The probe and methods used to map fields have been previouslydescribed (Bordelon et al., 2011; Bordelon et al., 2012). The fieldamplitude was measured in the coil center before each set of trials. Themeasured amplitude in this point is reported as the experimentalamplitude.

Water Jacket

Custom built water is jacket described elsewhere (Kumar et al., 2013),briefly, water jacket was constructed from concentric poly-acrylictubing filled with distilled water. To make the water cage device, twoholes were drilled into the tube at opposite ends. Acrylic hose adapterswere screwed into the holes with Teflon tape as a sealant. Water jacketinserted into the AMF coil can be seen in the FIG. 10. The water jackethelps anesthetized small animals to maintain the body temperature closeto physiological range when exposed to AMF amplitudes <90 kA/m (Kumar etal., In Press).

Temperature Measurements

Anesthetized mouse was directly injected with JHU-MIONs. Intratumoral,rectal and contralateral skin temperatures were measured with threeRF-resistant fiber optic temperature probes (FISO, Inc., Quebec,Canada). Animal with the fiber optic temperature probes was placed in afashioned 50 mL conical tube and inserted into the water jacket.Temperatures were recorded at one-second intervals.

Example 4 Rectangular Induction Coil Field Uniformity

AMF amplitude map from the measured data is shown in the FIG. 10. Fieldwas found to be uniform (within±10%) in 10 cm (x-axis)×14 cm (y-axis)area. The 48-well plate used in the study has outer dimensions of 8.6cm×12.8 cm, lower surface area than the measured uniform field surfacearea. All the wells in the in-vitro experiments are exposed to uniformAMF amplitude. The results are displayed in the plot of FIG. 10 agreesvery well with the modeling results previously reported (Nemkov et al.,2011).

Thermal Correction

The probes were initially immersed together in close contact in a warmwater bath at a temperature close to the target temperature of thecooling plate. The temperatures were averaged over a stable timeinterval to identify offsets in their temperature measurements. Thecooling plate temperature was then measured in the four quadrants of theplate at relevant voltage settings. The offset values were thensubtracted from the recorded data to form corrected data.

Example 5 In Vitro Experiments Temperature Measurements

Temperature data for control (cell only) and three IONPs (BNFs, SPIOsand JHU-MIONs) are shown in FIG. 11. Recorded temperatures showedsignificant heating due to JHU-MIONs compared to control (cell only),BNFs and SPIOs. For JHU-MIONs the recorded temperature showed a maximumtemperature rise of ˜7° C. while control, BNFs and SPIOs the temperaturerise was ≤3° C.

Cell Viability

Consistent with temperature measurement data DU145 cells treated athigher concentrations JHU-MIONs (2-3 mg/mL Fe) show the lowestviabilities as compared to BNFs and SPIOs. Data shown in the FIG. 12 arenormalized to the control without IONPs. Particles alone had noobservable effect on the cell viability.

Example 6 In Vivo Experiments

FIG. 13 shows the temperature versus treatment time for intratumoral,contralateral and rectal temperatures. Intratumoral temperature rise of˜11° C. was observed. A net temperature rise of ˜4° C. was observed inthe rectal temperature.

Example 7 Discussion of In Vitro and In Vivo Experiments

Feasibility of using previously reported rectangular induction coilcapable generating a uniform field (10 cm×14 cm) for in-vitroexperiments was shown for the first time in this study. Recordedtemperatures demonstrate JHU-MIONs heating ability compared to BNFs andSPIOs. Measured temperatures are relative measurements based uponcomparison with controls, and do not represent absolute measures oftemperature experienced by the cells. The measured temperatures providea relative means to compare among particles. As result of the higherheat generated by the JHU-MIONs at low AMF amplitude (20 kA/m), theDU145 cell viability was found to be significantly reduced compared toBNFs (at 2-3 mg Fe/mL, FIG. 13).

During MNH, animals are anesthetized, whereupon the animals becomehypothermic because normal thermoregulatory controls are temporarilycompromised (Adair and Black, 2003; Black, 2006; Ivkov et al., 2005).Heat loss occurs through respiration and body surface cooling if ambienttemperatures are significantly lower than the physiological norm. AMFdeposits non-specific heat, potentially creating significant thermalgradients throughout the animal, which could be detrimental. During theinitial phase of the MNH treatment, animal losses heat under theinfluence of anesthesia. After prolonged exposure to AMF additional heatis deposited in to the animal. If active and homogenizing temperaturecontrol can be provided during mNPH, safety may improve allowingincreased limits on maximum allowable H-values (Kumar et al., 2013).Previous mouse studies suggest that rectal temperature change(ΔTRectal), should be maintained below 5° C., or below 42° C., for thesafety of the animal (Ivkov et al., 2005). Here it is shown that in oneembodiment, an ideal setting of 29 KA/m and ˜4 mg Fe/g of tumor, AMFtreatment yielded ΔTTumor>10° C. while maintaining the ΔTRectal<5° C.Both AMF amplitude and amount of iron injected are lower compared with43.8 kA/m and 5 mg Fe/g of tumor used in Dennis et al., 2009 to achievemaximum ΔTTumor˜10° C.⁹. In the same study, three out of four animalswith mean ΔTTumor˜15° C.@55.7 kA/m and 5 mg Fe/g of tumor) showedcomplete regression of tumors after 15 min treatment. Considering lowamounts of iron and AMF amplitudes used in this study JHU-MIONs have thepotential to translate into a clinical product. Preclinical studiesusing JHU-MIONs are underway.

In the present study, the therapeutic relevant heating characteristicsof the JHU-MIONs at low AMF amplitudes (20 kA/m) in an in-vitro set-uphave been demonstrated. In-vivo feasibility test of JHU-MIONsdemonstrated their ability to rise intratumoral temperatureapproximately 11° C. when injected with iron concentrations <4 mg Fe/gof tumor and exposed to an AMF amplitude of 29 kA/m. These preliminarystudies provide motivation for further research and preclinicaldevelopment of the JHU-MIONS.

Example 8 Materials and Measurement Methods for Amplitude-DependentEffect Experiments

Particle size analysis by DLS, Transmission Electron Microscope (TEM)and Scanning Electron Microscope (SEM) methods, x-ray diffraction (XRD)analysis, iron concentration measurements by ICP-AES, and specificabsorption rate measurements were performed as described in Example 1.

Mössbauer Spectroscopy

The composition of the samples was determined by Fe⁵⁷ TransmissionMossbauer spectroscopy using a constant acceleration spectrometercalibrated with α-Fe at room temperature and a 1 GBq Co⁵⁷ source.Spectra were collected with the samples in a Janis SHI-850 closed cyclerefrigeration system (Janis Research Co., Wilmington, Mass.) at 10K.

Magnetometry

Hysteresis loops were measured at temperatures ranging between 300K to5K from ±5570 kA/m (±70,000 Oe) using a Superconducting QuantumInterference Device Vibrating Sample Magnetometer (SQUID VSM) fromQuantum Design, Inc. (San Diego, Calif.). The colloidal samples wereloaded into Kel-F liquid capsules from LakeShore Cryogenics(Westerville, Ohio), and sealed with epoxy to prevent evaporation of thewater solvent during measurement under vacuum.

SANS

Unpolarized SANS data taken with 0.84 nm wavelength neutrons intransmission using three detector settings in order to span the range ofscattering vectors Q from 3×10⁻⁵ to 5×10⁻¹ Å⁻¹.

For PASANS, to cover the necessary Q range of (0.005-0.2) Å⁻¹ for theBNFs, two different wavelengths of neutrons were used: (5±0.6) A and(7.5±0.9) Å. The 5 Å (7.5 Å) neutrons were polarized with an efficiencyof 0.888±0.005 (0.935±0.003) by scattering from an FeSi supermirror,sending the spin-up (+) neutrons down the beam line. Prior tointeraction with the sample, the incident neutron polarization directioncan be reversed at any time using an electromagnetic flipper coil with aflipping efficiency of 0.988±0.004 (0.979±0.003). [For the JHUs andSPIOs, only the (7.5±0.8) A neutrons were used to cover the necessary Qrange of (0.005-0.06) Å⁻¹. The 7.5 Å neutrons were polarized with anefficiency of 0.95±0.02 with the FeSi supermirror, and theelectromagnetic flipper coil had a flipping efficiency of 0.975±0.009.]After scattering from the sample of interest, an analyzing glass cellfilled with polarized ³He gas preferentially allows neutrons with spinsaligned parallel to the ³He atoms to pass through, while absorbingneutrons of the other spin state. The orientation of the ³He spin filtercan also be reversed at any time with a nuclear magnetic resonance pulseof an appropriate frequency. The data is then corrected for detectorefficiency, background, and the polarization efficiency plus thetime-dependence of the ³He cell according to previously describedmethods.

Example 9 Amplitude-Dependent Effect Experiments

With the development of new syntheses for controlling size, shape, andcrystallinity of magnetic nanoparticles in the last two decades, newapplications of magnetic nanoparticles have been developed. Inparticular, the biomedical applications of magnetic nanoparticles havebeen rapidly expanding from ex vivo diagnostic tools like cell- (Shao etal., 2012) and immuno-assays (Jin et al., 2009), to in vivo diagnostictools like magnetic resonance imaging (MRI; Swierczewska et al., 2011)and magnetic particle imaging (MPI; Gleich and Weizenecker, 2005), aswell as to therapeutic techniques such as hyperthermia (Lehmann et al.,2008) and drug delivery (Kim et al., 2008). However, in developing anyparticular nanoparticle system for a specific application, there is acomplex interplay between the structure and the optimal magneticproperties. This interplay is significantly constrained oncebiocompatibility (composition, stability in blood, bio-distribution,etc.) are added.

In hyperthermia, these constraints are particularly evident. Themagnetic nanoparticles must have a large enough moment to interact, butnot so large that the magnetic attraction is stronger thansteric/electrostatic repulsion; otherwise, the nanoparticles willagglomerate. The nanoparticles must also have a significant anisotropypresent, but they cannot have an aspect ratio greater than approximately3:1 or their circulation time in the bloodstream will be very short. Themagnetic moment of the core material must be maximized, but they mustalso be stable under physiological conditions and biocompatible. Theseand other constraints leave a narrow window in which to design effectivenanoparticles for hyperthermia.

Finally, the methodology itself also imposes constraints. Inhyperthermia, the magnetic nanoparticles are subjected to an alternatingmagnetic field in order to deposit energy in the form of heat into thesurrounding cancerous tissue. However, the amount of energy deposited,as quantified by specific absorption rate (SAR) or, more correctly, thespecific loss power (SLP), is determined, in part, by the magnetic fieldamplitude. The magnetic field amplitude, in turn, is limited by thepower generation requirements to produce a uniform field over for wholebody vs. localized regions. Therefore, it is critical to understand theparameters important in determining how a given magnetic nanoparticlesystem responds to the magnetic field amplitude at fixed frequency. Thisis especially important, because most experimental systems only examinethe SLP at one or two fields at fixed frequency. To date, no systematicstudies have been performed that look at the behavior of a nanoparticlesystem as a function of field or frequency.

In this Example, the heat characteristics are examined of threedifferent nanoparticle systems, which are all similar in size andcomposition, as a function of applied field at fixed frequency. Aftercharacterizing the nanoparticles both physically and magnetically, theparameters responsible for determining their heat generation areidentified. In addition to the previously established importance ofinteractions (Dennis et al., 2009), it has been found that the internalstructure of the magnetic nanoparticles plays a significant role indetermining the SLP.

The first system, called Bionized Nano-Ferrite (BNFs), is synthesized bya high temperature, high pressure homogenization process (Gruettner etal., 2007). As determined by Mossbauer spectroscopy (FIG. 17), theresulting core is composed of 77% Fe₃O₄ and 18% γ-Fe₂O₃ with about 4%Fe(OH)₂. Unpolarized Small Angle Neutron Scattering (SANS) demonstratesthat the core is composed of parallelepipeds approximately 8.8 nm×24nm×96 nm (data not shown). Scherrer analysis from X-Ray Diffraction(XRD) confirms that the core is composed of grains which are (12±2) nmin size, on the order of the smallest dimension of the parallelepipeds(data not shown). The core is coated twice with a 40,000 Dalton dextranshell. Dynamic Light Scattering (DLS) indicates that the total particlediameter is 126 nm with a distribution (σ) of 39% (FIG. 14).Transmission Electron Microscopy (TEM) demonstrates that the iron oxidecrystallites are ˜20 nm, in agreement with SANS (data not shown). Forthe subsequent magnetometry and SANS measurements, unless otherwiseindicated, the samples are measured in water at a particle (iron)concentration of 22 (11) mg/mL.

The second system, called JI-IU particles, is synthesized by ahigh-gravity controlled precipitation method. As determined by Mossbauerspectroscopy (FIG. 18), the resulting core is composed of 76% Fe₃O₄ and24% γ-Fe₂O₃. Unpolarized SANS (data not shown) demonstrates that thecore is composed of grains which are approximately spherical in shapeand (8±3) nm in radius. Scherrer analysis from XRD (data not shown)confirms that the core is composed of grains which are (9±1) nm in size.The core is coated with a dextran shell, and DLS confirms a hydrodynamicdiameter of 117 nm with a distribution (σ) of 41% (FIG. 15). TEMdemonstrate that the iron oxide crystallites are ˜15 nm, in agreementwith SANS (data not shown). For the subsequent magnetometry and SANSmeasurements, unless otherwise indicated, the samples are measured inwater at a particle (iron) concentration of 64 (9.2) mg/mL.

The third system, called Nanomag-SPIOs, is synthesized by aco-precipitation of iron salts in the presence of dextran (Rudershausenet al., 2002). As determined by Mossbauer spectroscopy (FIG. 19), theresulting nanocrystallites are composed of 87% Fe₃O₄ and 13% γ-Fe₂O₃.Unpolarized SANS (data not shown) demonstrates that the core is composedof grains which are approximately spherical in shape and, on average,4.4 nm in radius. Scherrer analysis from XRD confirms that thenanocrystallites are composed of grains which are ˜8 nm in size (datanot shown). The nanocrystallites are embedded within a 40,000 Daltondextran shell, and DLS confirms a hydrodynamic diameter of 106 nm with adistribution (σ) of 50% (FIG. 16). TEM demonstrate that thenanocrystallites are ˜20 nm, demonstrating the presence of agglomerationof the grains (data not shown). For the subsequent magnetometry and SANSmeasurements, unless otherwise indicated, the samples are measured inwater at a particle (iron) concentration of 96 (12.5) mg/mL.

The saturation magnetizations (MS) of the particles were measured atroom temperature using SQUID magnetometry and normalized to the ironcontent for direct comparison with the SLP (FIG. 20). (For comparisonwith the literature, the saturation magnetization is also normalized tototal particle mass, including dextran, and shown in brackets. Due tothe inclusion of the mass of the dextran, these saturation values aresignificantly below that of the bulk.) For the BNFs, MS is the highestat (80.76±0.06) A-m2/kg [(19.86±0.02) A-m2/kg]. For the JHUs,MS=(73.6±0.1) A-m2/kg [(10.558±0.001) A-m2/kg]. For the SPIOs, MS is thelowest at (67.69±0.01) A-m2/kg [(8.814±0.001) A-m2/kg]. In summary, MSwhen normalized to iron content, only varies by 15% across the threesamples. However, upon consideration of the region about zero field withrealistic hyperthermia field amplitudes (±80 kA/m), there are smalldifferences in the shape of the hysteresis loops (inset to FIG. 20).These can be most readily attributed to either interactions (Taketomiand Shull, 2002) in the sample or differences in the anisotropy (Poddaret al., 2008). The interactions can be probed by looking at the virgincurve and comparing it with the major loop. The BNFs exhibit stronginteractions (shown by the deviations from the major loop), while theJHU particles have somewhat weaker interactions, and the SPIOs have veryweak interactions. The anisotropy field (Hk) can be directly measuredusing transverse susceptibility (data not shown). (Due to signal tonoise issues, these measurements are made at 5K.) For the BNFs, μ0Hk=10mT. For the JHUs, μ0Hk=14 mT. Given that μ0Hk=2 Keff/Ms,Keff=2.977(3)×10-7 J and 2.82(2)×10-7 J for the BNFs and JHUs,respectively. (Normalized to iron concentration, Keff=4.034(4)×10-4 J/kgof Fe and 5.11(4)×10-4 J/kg of Fe for the BNFs and JHUs, respectively.)So, magnetically, the nanoparticles are all similar. Therefore, onlysmall variations in the SLP are expected between the different samples.In contrast, there is a significant variation (FIG. 21) in the SLP ofthe samples, both in magnitude as well as onset and slope, as a functionof the applied magnetic field. (The measurement frequency is fixed at150 kHz.) The BNFs fail to generate significant heat until approximately20 kA/m, and the SLP is still increasing past 500 W/g-Fe at 65 kA/m. TheJHUs start to generate significant heat above approximately 5 kA/m, andthen plateau at about 440 W/g-Fe at approximately 50 kA/m. The SPIOsplateau at about 150 W/g-Fe at approximately 30 kA/m. These significantvariations, however, cannot originate from the minor differences seen sofar. In particular, they cannot be accounted for simply by changes inthe magnetization with field, as can be clearly demonstrated byconsidering the JHU and BNF nanoparticles above 50 kA/m. Here, when theJHUs are saturating in SLP, the DC hysteresis loop of the JHUs areoverlapping with the BNFs, and both are still increasing. Therefore,there must be an additional factor which has not been accounted for yet.

In an AC magnetic field, such as is applied during hyperthermia, themoments have to very rapidly reorient themselves to align with thefield. Therefore, the internal magnetic structure may play a criticalrole in both the mechanism of reorientation as well as the energy thatmust be provided for this reorientation. To explore the detailedinternal magnetic structure, polarization analyzed small angle neutronscattering (PASANS) was used at room temperature on the nanoparticles inD₂O under an applied guide magnetic field of 1.5 mT. (Unpolarizedneutrons were not sufficient for determining the magnetic structure,since the magnetic signal is relatively weak when the nanoparticles arecontrasted with H₂O, and the magnetic structure determination isambiguous when the nanoparticles are contrasted with D₂O.) The SANSmeasurements were performed at the NIST Center for Neutron Research onthe NG-3 beamline.

For the BNFs, to cover the necessary Q range of (0.005-0.2) Å⁻¹, twodifferent wavelengths of neutrons were used: (5±0.6) Å and (7.5±0.9) Å.The 5 Å (7.5 Å) neutrons were polarized with an efficiency of0.888±0.005 (0.935±0.003) by scattering from an FeSi supermirror,sending the spin-up (+) neutrons down the beam line. Prior tointeraction with the sample, the incident neutron polarization directioncan be reversed at any time using an electromagnetic flipper coil with aflipping efficiency of 0.988±0.004 (0.979±0.003). [For the JHUs andSPIOs, only the (7.5±0.8) A neutrons were used to cover the necessary Qrange of (0.005-0.06) Å⁻¹. The 7.5 Å neutrons were polarized with anefficiency of 0.95±0.02 with the FeSi supermirror, and theelectromagnetic flipper coil had a flipping efficiency of 0.975±0.009.]After scattering from the sample of interest, an analyzing glass cellfilled with polarized 3He gas preferentially allows neutrons with spinsaligned parallel to the 3He atoms to pass through, while absorbingneutrons of the other spin state. The orientation of the 3He spin filtercan also be reversed at any time with a nuclear magnetic resonance pulseof an appropriate frequency. The data is then corrected for detectorefficiency, background, and the polarization efficiency plus thetime-dependence of the 3He cell according to previously describedmethods.

Starting with the neutron polarization spin state as + or −, measurementof all four neutron spin cross-sections (++, +−, −+, and −−) allows forthe unique separation of nuclear scattering (N2) from magneticscattering, irrespective of whether the sample is magneticallysaturated. Simply, the “+ to +” and “− to −” scattering (ornon-spin-flip scattering) contains information about primarily magneticscattering from moments parallel to the applied sample field and nuclearscattering, while “+ to −” and “− to +” (or spin-flip scattering)contains only magnetic scattering. The complete, angle-dependentpolarization selection rules simplify at several key angles and enablethe unambiguous separation of the nuclear scattering (N2) from magneticscattering of moments parallel to the applied guide field (M2Y=M2PARL)and those perpendicular to the applied field (M2PERP which is a linearcombination of M2X and M2Z) for any field ≥1 mT. Starting from theefficiency-corrected 2D scattering patterns from the magnetitenanoparticles in the 1.5 mT field parallel to the Y axis,area-normalized sector slices of ±10 o are taken about θ=0 o, where θ isthe angle between the X axis (horizontal midline of the detector) andthe projection of Q onto the X-Y detector plane. At this specific angle,M2PERP=M2Z.

Once the three components, N2, M2PERP, and M2PARL, have been calculated,they can be fit to the appropriate model to (a) verify the nuclearstructure determined previously with unpolarized SANS and (b) determinethe magnetic domain sizes parallel and perpendicular to the appliedmagnetic field of 1.5 mT.

In terms of the BNFs, fitting N2 with the parallelepiped model resultsin 7 nm×33 nm×77 nm parallelepipeds which are similar to thosedetermined from the unpolarized neutrons (FIG. 22). The magneticscattering shows dramatically different sizes parallel and perpendicularto the applied magnetic field (FIG. 23). This data was fit with aparallelepiped model, but a spherical model also works as the fit ismost sensitive to the smallest dimension. This results in a domainstructure which is ˜22 nm parallel to the field and ˜8 nm perpendicularto the field, and is shown graphically in the inset to FIG. 23. Asdetermined previously (Krycka et al., 2011), this magnetic domainstructure does not necessarily align with the crystallographic grains.This break-up into domains is not unsurprising, although the core sizeis smaller than the nominal single domain size for magnetite.Furthermore, given the measured parameters, this structure is inagreement with micromagnetic simulations.

For the JHUs, N2 still fits nicely to the spherical model, with the samegrain size as determined with the unpolarized beam (FIG. 22). Themagnetic scattering also demonstrates a domain structure, but theaverage domain size is significantly larger than what is seen in theBNFs (FIG. 24). This data was fit with a spherical model, which resultsin a domain structure which is ˜19 nm in radius parallel to the fieldand ˜6 nm in radius perpendicular. These sizes indicates that, for a 50nm core, the particles are magnetically more core/shell, where the coreis 38 nm in diameter and magnetized parallel to the field, and theshell, of thickness 6 nm, is magnetized perpendicular to the field. Thisis shown graphically in the inset to FIG. 24.

For the SPIOs, N2 still fits nicely to the spherical model, with thesame grain size (within error) as determined with the unpolarized beam(FIG. 22). In contrast, the magnetic scattering parallel to the field isbarely above background (FIG. 25). Plots of the subtracted signal, anindication of the net moment, are so low that effectively there is nomeasureable magnetic moment. Therefore, it is reasonable to assume thatthe domain size parallel to the field is commensurate with the nucleargrain size. (It is possible to plot a curve through the M2PARL databased on this assumption.) The magnetic scattering perpendicular to thefield is slightly better (FIG. 25), and clearly shows a domain size of 6nm in radius, which is commensurate with the nuclear grain size. Thisdomain structure is shown graphically in the inset to FIG. 25.

In summary, the BNFs have a high saturation magnetization, with a momentthat breaks up into long slender domains. Without wishing to be bound toany one particular theory, it is believed that the 2% iron hydroxidecomponent might be a thin surface coating on each parallelepiped thatforms prior to the final core formation. (There is no known method ofverifying absolutely that this is the case, because TEM does not havethe resolution to identify the differences over a single monolayer.)However, this means that each domain will be strongly coupled to itsneighbor. Therefore, a larger field must be applied to change thatcoupling and force all of the moments to align. This accounts for thehigher field required before on-set of significant heating.

In contrast, the JHUs have a slightly lower saturation magnetization (by˜9%) than the BNFs, and break up into one or two large domains. The JHUsalso have a larger anisotropy (by ˜20%), perhaps controlled by thehigher concentration of γ-Fe₂O₃, which in bulk has a ˜50% largeranisotropy than that of Fe₃O₄. We also hypothesize that duringformation, each grain starts out as Fe₃O₄, with the surface changing toγ-Fe₂O₃ during the high temperature phase. Eventually, these grainscoalesce to form the final core, but the individual grains have an Fe₃O₄core and γ-Fe₂O₃ shell. The nominal reduction in the saturationmagnetization of bulk γ-Fe₂O₃ shell in combination with the change inanisotropy, could allow the grains to be less coupled togethermagnetically than the BNFs. This would result in a lower on-set ofsignificant SLP, than the BNFs.

Finally, the lack of coherent moment present in the SPIOs means that allof the individual grains are weakly coupled, at best. Therefore, theyhave the lowest on-set of measurable SLP, because it is easier tomagnetize each individual grain, since the total moment is beingmagnetized, and multiple domains do not have to be aligned. However, dueto their weak interactions, they are not capable of producing nearly asmuch heat.

This means that when designing magnetic nanoparticles for hyperthermia,the internal magnetic structure and the exchange coupling between anydomains must be taken into account as well as any coupling(interactions) between nanoparticles and the resulting anisotropy. Thiswill allow for design of particles at a fixed frequency to maximizeheating at lower fields for whole body or metastatic cancers versusbeing able to take advantage of higher fields and therefore higher SLPfrom localized cancers like neck and throat or prostate. However, it isexpected that when changing the frequency, there will be optimalinteraction strength both between particles and within a particle tomaximize the heating. At higher frequencies, such as 1 MHz (Southern etal.), strong interactions will slow down the dynamic response of theparticles, thereby requiring higher fields to flip the magnetization orresulting in lower SLP for a given field.

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

Adair, E. R., and Black, D. R., “Thermoregulatory responses to RF energyabsorption,” Bioelectromagnetics 6(Supplement), S17-S38 (2003);

Atkinson, W. J., Brezovich, I. A., Chakraborty, D. P., “Usablefrequencies in hyperthermia with thermal seeds,” IEEE Trans. Biomed.Eng. 31, 70-75 (1984);

Black, D. R., [Thermoregulation in the presence of radio frequencyfields], Biological and Medical Aspects of Electromagnetic Fields, 3rdEdition, Boca Raton, 215-226 (2006);

Bordelon, D., et al. “Magnetic nanoparticle heating efficiency revealsmagneto-structural differences when characterized with wide ranging andhigh amplitude alternating magnetic fields,” Journal of Applied Physics109, 12904.1-12904.8 (2011);

Bordelon, D., Goldstein, R., Nemkov, V., Kumar, A., Jackowski, J.,DeWeese, T. L., Ivkov, R., “Modified solenoid coil that efficientlyproduces high amplitude AC magnetic fields with enhanced uniformity forbiomedical applications,” IEEE Trans. on Magnetics 48, 47-52 (2012);

Dennis C L, et al. “Nearly complete regression of tumors via collectivebehavior of magnetic nanoparticles in hyperthermia,” Nanotechnology20(39), Article Number 395103 (2009);

Dennis C. L., A. J. Jackson, J. A. Borchers, P. J. Hoopes, R.Strawbridge, A. R. Foreman, J. van Lierop, C. Grüttner, and R. Ivkov,Nanotechnology, 20 (2009) 395103;

Gruettner C, K. Mueller, J. Teller, F. Westphal, A. Foreman, and R.Ivkov, J. “Synthesis and antibody conjugation of magnetic nanoparticleswith improved specific power absorption rates for alternating magneticfield cancer therapy,” Journal of Magnetism and Magnetic Materials311(1), 181-186 (2007);

C. Grüttner, J. Teller, W. Schutt, F. Westphal, C. Schümichen and B. R.Paulke, Preparation and Characterization of Magnetic Nanospheres for invivo Application. In Scientific and Clinical Application of MagneticCarriers (U. O. Hafeli, W. Schutt, J. Teller and M. Zborowski, Eds.),pp. 53-68. Plenum Press, New York, 1997;

Hedayati, M., Thomas, O., Abubaker-Sharif, B., Zhou, H., Cornejo, C.,Zhang, Y., Wabler, M., Mihalic, J., Gruettner, C., Westphal, F., Geyh,A., Deweese, T. L., Ivkov, R., “The effect of cell cluster size onintracellular nanoparticle-mediated hyperthermia: is it possible totreat microscopic tumors?,” Nanomedicine (Lond) 8(1), 29-41 (2013);

Ivkov, R., DeNardo, S. J., Daum, W., Foreman, A. R., Goldstein, R. C.,Nemkov, V. S., DeNardo, G. L., “Application of high amplitudealternating magnetic fields for heat induction of nanoparticleslocalized in cancer,” Clin. Cancer Res. 11(19 Suppl), 7093s-7103s(2005);

Jordan, A., Wust. P., Scholz, R., Faehling, H., Krause, J. and Felix,R., [Magnetic Fluid Hyperthermia (MFH)], Scientific and ClinicalApplications of Magnetic Carriers, New York, 569-595 (1997);

Kim, J., J. E. Lee, S. H. Lee, J. H. Yu, J. H. Lee, T. G. Park, and T.Hyeon, Adv. Mater., 20 (2008) 478;

Krycka, K. L., A. J. Jackson, J. A. Borchers, J. Shih, R. Briber, R.Ivkov, C. Gratner, and C. L. Dennis, Journal of Applied Physics, 109(2011) 07B513.

Kumar, A., Attaluri, A., Mallipudi, R, Cornejo, C., Bordelon, D.,Armour, M., Morua, K., DeWeese, T. L., Ivkov, R., “Method to reducenon-specific tissue heating of small animals in solenoid coils,” Int. JHyperthermia, 29, 106-120 (2013);

Nemkov V, et al. “Magnetic field generating inductor for cancerhyperthermia research,” Compel 10(5), 1626-1636 (2011);

Poddar, P., M. B. Morales, N. A. Frey, S. A. Morrison, E. E. Carpenter,and H. Srikanth, J. Appl. Phys., 104 (2008);

Repetto G, et al. “Neutral red uptake assay for the estimation of cellviability/cytotoxicity,” Nature Protocols 3(7), 1125-1131 (2008);

Rosensweig, R E., “Heating magnetic fluid with alternating magneticfield,” J. Magnetism and Magn. Materials 252, 370-374 (2002);

Rudershausen S, Grüttner C, Frank M, Teller J, Westphal F:Multifunctional Superparamagnetic Nanoparticles for Life ScienceApplications. European Cell and Materials 3, 81-83 (2002);

Southern, P., D. Ortega, C. Johansson, and Q. Pankhurst, Talk 35 of the9th International Conference on the Scientific and Clinical Applicationsof Magnetic Carriers, Minneapolis, Minn.; and

Taketomi, S. and R. D. Shull, J. Appl. Phys., 91 (2002) 8546-8548.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

1. A process for preparing one or more surfactant-coated magnetic metaloxide particles, the process comprising: (a) providing a salt solutionof a metal; (b) contacting the salt solution of the metal with aprecipitant solution to form a reactant solution; (c) rapidlymicro-mixing the reactant solution to initiate formation of metal oxidecrystals under controlled nucleation conditions; (d) continuing torapidly micro-mix the reactant solution under high gravity conditions tocontrol crystal growth of one or more metal oxide particles formedtherein; (e) coating the one or more metal oxide particles with asurfactant; (f) separating the one or more coated metal oxide particlesfrom the reactant solution and one or more by-products, if present,formed therein; and (g) exposing the one or more coated metal oxideparticles to high temperature and high pressure in an inert gasenvironment for a period of time to form one or more surfactant-coatedmagnetic metal oxide particles.
 2. The process of claim 1, wherein thereactant solution comprises an iron precursor solution comprisinganhydrous FeCl₃ and FeCl₂.4H₂O in hydrochloric acid.
 3. The process ofclaim 2, wherein the reactant solution further comprises ammonia.
 4. Theprocess of claim 1, wherein the coating comprises citric acid.
 5. Theprocess of claim 1, wherein the salt solution comprises a metal saltcomprising a metal selected from the group consisting of Fe, Co, Ni, andSm.
 6. The process of claim 5, wherein the metal salt comprises ananionic species selected from the group consisting of chloride, bromide,fluoride, iodide, nitrate (NO₃), sulfate (SO₄), chlorate (ClO₄), andphosphate (PO₄).
 7. The process of claim 1, wherein the precipitantsolution comprises at least one member selected from the groupconsisting of NaOH, ammonium hydroxide (NH₄OH), and another hydroxide ofGroup I or II elements from the Periodic Table of elements.
 8. Theprocess of claim 1, wherein the reactant solution comprises at least onemember selected from the group consisting of a hydroxide, a carbonate,and a phosphate.
 9. The process of claim 1, wherein the surfactant isselected from the group consisting of an organic acid, a lipid, aphospholipid, an oleate, an ester, a sulfate, a diol, and a polymer. 10.The process of claim 1, wherein the exposing of the one or more coatedmetal oxide particles to high temperature and high pressure is conductedat about 130° C. for about 5 hours.
 11. The process of claim 1, whereinthe pressure range is from about 1 atmosphere to about 1,000atmospheres.
 12. One or more surfactant-coated magnetic metal oxideparticles prepared by the method of claim
 1. 13. The one or moresurfactant-coated magnetic metal oxide particles of claim 12, whereinthe particles have a substantially isotopic shape.
 14. The one or moresurfactant-coated magnetic metal oxide particles of claim 12, whereinthe particles have a dimension ranging from about 30 nm to about 100 nm.15. The one or more surfactant-coated magnetic metal oxide particles ofclaim 12, wherein the particles comprise about 76% Fe₃O₄ and about 24%γ-Fe₂O₃.
 16. The one or more surfactant-coated magnetic metal oxideparticles of claim 12, wherein the particles are substantially free ofFe(OH)₂.
 17. A magnetic metal oxide nanoparticle prepared from ahigh-gravity controlled precipitation reaction, the nanoparticlecomprising: (a) iron oxide crystals having a dimension ranging fromabout 5 nm to about 100 nm; and (b) a surfactant coating; wherein thenanoparticle has a heating property of greater than about 60 W/g Fe inan alternating current (AC) magnetic field having a frequency of rangingfrom about 50 kHz and to about 1 MHz and an amplitude ranging from about0.080 kA/m to about 80 kA/m.
 18. A biocompatible suspension comprising amagnetic metal oxide nanoparticle of claim 12 and water.
 19. A methodfor treating a diseased tissue, the method comprising: (a) administeringto a tissue or a subject in need of treatment thereof, a therapeuticallyeffective amount of a magnetic nanoparticle comprising surfactant-coatediron oxide crystals prepared from a high-gravity controlledprecipitation process; and (b) subjecting the tissue or subject, or aportion of the tissue or subject to an alternating current (AC) magneticfield having frequency ranging from about 50 kHz to about 1 MHz andhaving an amplitude (peak-to-peak) ranging from about 0.080 kA/m toabout 50 kA/m.
 20. The method of claim 19, wherein the diseased tissuecomprises a cancer tissue.
 21. (canceled)
 22. (canceled)
 23. A kit fortreating a diseased tissue, the kit comprising a magnetic metal oxidenanoparticle of claim 12.